MicroRNAs (miRNAs) are small noncoding RNAs that regulate eukaryotic gene expression by binding to regions of imperfect complementarity in mRNAs, typically in the 3′ UTR, recruiting an Argonaute (Ago) protein complex that usually results in translational repression or destabilization of the target RNA. The translation and decay of mRNAs are closely linked, competing processes, and whether the miRNA-induced silencing complex (RISC) acts primarily to reduce translation or stability of the mRNA remains controversial. miR-122 is an abundant, liver-specific miRNA that is an unusual host factor for hepatitis C virus (HCV), an important cause of liver disease in humans. Prior studies show that it binds the 5′ UTR of the messenger-sense HCV RNA genome, stimulating translation and promoting genome replication by an unknown mechanism. Here we show that miR-122 binds HCV RNA in association with Ago2 and that this slows decay of the viral genome in infected cells. The stabilizing action of miR-122 does not require the viral RNA to be translationally active nor engaged in replication, and can be functionally substituted by a nonmethylated 5′ cap. Our data demonstrate that a RISC-like complex mediates the stability of HCV RNA and suggest that Ago2 and miR-122 act coordinately to protect the viral genome from 5′ exonuclease activity of the host mRNA decay machinery. miR-122 thus acts in an unconventional fashion to stabilize HCV RNA and slow its decay, expanding the repertoire of mechanisms by which miRNAs modulate gene expression.

A cloverleaf structure at the 5' terminus of poliovirus RNA binds viral and cellular proteins. To examine the role of the cloverleaf in poliovirus replication, we determined how cloverleaf mutations affected the stability, translation and replication of poliovirus RNA in HeLa S10 translation-replication reactions. Mutations within the cloverleaf destabilized viral RNA in these reactions. Adding a 5' 7-methyl guanosine cap fully restored the stability of the mutant RNAs and had no effect on their translation. These results indicate that the 5' cloverleaf normally protects uncapped poliovirus RNA from rapid degradation by cellular nucleases. Preinitiation RNA replication complexes formed with the capped mutant RNAs were used to measure negative-strand synthesis. Although the mutant RNAs were stable and functional mRNAs, they were not active templates for negative-strand RNA synthesis. Therefore, the 5' cloverleaf is a multifunctional cis-acting replication element required for the initiation of negative-strand RNA synthesis. We propose a replication model in which the 5' and 3' ends of viral RNA interact to form a circular ribonucleoprotein complex that regulates the stability, translation and replication of poliovirus RNA.

We previously showed that a noncoding subgenomic flavivirus RNA (sfRNA) is required for viral pathogenicity, as a mutant West Nile virus (WNV) deficient in sfRNA production replicated poorly in wild-type mice. To investigate the possible immunomodulatory or immune evasive functions of sfRNA, we utilized mice and cells deficient in elements of the type I interferon (IFN) response. Replication of the sfRNA mutant WNV was rescued in mice and cells lacking interferon regulatory factor 3 (IRF-3) and IRF-7 and in mice lacking the type I alpha/beta interferon receptor (IFNAR), suggesting a contribution for sfRNA in overcoming the antiviral response mediated by type I IFN. This was confirmed by demonstrating rescue of mutant virus replication in the presence of IFNAR neutralizing antibodies, greater sensitivity of mutant virus replication to IFN-α pretreatment, partial rescue of its infectivity in cells deficient in RNase L, and direct effects of transfected sfRNA on rescuing replication of unrelated Semliki Forest virus in cells pretreated with IFN-α. The results define a novel function of sfRNA in flavivirus pathogenesis via its contribution to viral evasion of the type I interferon response.

The cis-acting replication element (CRE) is a 61-nucleotide stem-loop RNA structure found within the coding sequence of poliovirus protein 2C. Although the CRE is required for viral RNA replication, its precise role(s) in negative-and positive-strand RNA synthesis has not been defined. Adenosine in the loop of the CRE RNA structure functions as the template for the uridylylation of the viral protein VPg. VPgpUpU OH , the predominant product of CRE-dependent VPg uridylylation, is a putative primer for the poliovirus RNA-dependent RNA polymerase. By examining the sequential synthesis of negative-and positive-strand RNAs within preinitiation RNA replication complexes, we found that mutations that disrupt the structure of the CRE prevent VPg uridylylation and positive-strand RNA synthesis. The CRE mutations that inhibited the synthesis of VPgpUpU OH , however, did not inhibit negative-strand RNA synthesis. A Y3F mutation in VPg inhibited both VPgpUpU OH synthesis and negative-strand RNA synthesis, confirming the critical role of the tyrosine hydroxyl of VPg in VPg uridylylation and negative-strand RNA synthesis. trans-replication experiments demonstrated that the CRE and VPgpUpU OH were not required in cis or in trans for poliovirus negative-strand RNA synthesis. Because these results are inconsistent with existing models of poliovirus RNA replication, we propose a new four-step model that explains the roles of VPg, the CRE, and VPgpUpU OH in the asymmetric replication of poliovirus RNA.Poliovirus possesses a single-stranded positive-polarity RNA genome that is 7,441 nucleotides (nt) in length (39). VPg, a virally encoded 22-amino-acid protein, is covalently linked to the 5Ј uridine of poliovirus RNA. When released into the cytoplasm of susceptible cells, VPg is removed from the 5Ј terminus of poliovirus RNA by a host enzyme (1, 2). Following the removal of VPg, viral RNA functions sequentially as an mRNA for viral protein synthesis and then as a template for negative-strand RNA synthesis during viral RNA replication. Translation of the long open reading frame in viral mRNA yields a single polyprotein which undergoes both co-and posttranslational processing, culminating in the synthesis of viral capsid and replication proteins (21,24,32). The viral replication proteins interact with both the viral mRNA and components of the host cell to produce membrane-associated ribonucleoprotein complexes (9-11, 44, 45). cis-active RNA structures found within the 5Ј and 3Ј nontranslated regions (NTRs) of poliovirus RNA mediate the sequential translation and replication of viral RNA (4,8,20,28,38,41). Poliovirus mRNA, like other eukaryotic mRNAs (43), may circularize within messenger ribonucleoprotein complexes. Likewise, because viral negative-strand RNA synthesis requires RNA structures from both the 5Ј and 3Ј NTRs, the template for viral negative-strand RNA synthesis may exist in a circularized conformation via 20). Amplification of viral RNA within membranous replication complexes occurs asymmetrically. Viral RNA is first copi...

RNA recombination is important in the formation of picornavirus species groups and the ongoing evolution of viruses within species groups. In this study, we examined the structure and function of poliovirus polymerase, 3D pol , as it relates to RNA recombination. Recombination occurs when nascent RNA products exchange one viral RNA template for another during RNA replication. Because recombination is a natural aspect of picornavirus replication, we hypothesized that some features of 3D pol may exist, in part, to facilitate RNA recombination. Furthermore, we reasoned that alanine substitution mutations that disrupt 3D pol -RNA interactions within the polymerase elongation complex might increase and/or decrease the magnitudes of recombination. We found that an L420A mutation in 3D pol decreased the frequency of RNA recombination, whereas alanine substitutions at other sites in 3D pol increased the frequency of recombination. The 3D pol Leu420 side chain interacts with a ribose in the nascent RNA product 3 nucleotides from the active site of the polymerase. Notably, the L420A mutation that reduced recombination also rendered the virus more susceptible to inhibition by ribavirin, coincident with the accumulation of ribavirin-induced G¡A and C¡U mutations in viral RNA. We conclude that 3D pol Leu420 is critically important for RNA recombination and that RNA recombination contributes to ribavirin resistance. IMPORTANCE Recombination contributes to the formation of picornavirus species groups and the emergence of circulating vaccine-derived polioviruses (cVDPVs).The recombinant viruses that arise in nature are occasionally more fit than either parental strain, especially when the two partners in recombination are closely related, i.e., members of characteristic species groups, such as enterovirus species groups A to H or rhinovirus species groups A to C. Our study shows that RNA recombination requires conserved features of the viral polymerase. Furthermore, a polymerase mutation that disables recombination renders the virus more susceptible to the antiviral drug ribavirin, suggesting that recombination contributes to ribavirin resistance. Elucidating the molecular mechanisms of RNA replication and recombination may help mankind achieve and maintain poliovirus eradication. Picornaviruses are widespread in nature. Based on their ancient origins (1) and shared molecular features (2), picornaviruses are taxonomically organized by order, family, genus, species, and virus. Viruses in the family Picornaviridae are classified within 29 genera and 50 species groups. The genus Enterovirus, with 12 species groups (enterovirus species A to J and rhinovirus species A to C), contains the vast majority of medically important picornaviruses, including the polioviruses (PV), coxsackieviruses, enteroviruses, echoviruses, and rhinoviruses. These viruses, which possess single-stranded positive-sense RNA genomes, replicate via RNA intermediates in the cytoplasm of infected cells. A virus-encoded RNA-dependent RNA polymerase, 3D pol...

Triggering and propagating an intracellular innate immune response is essential for control of viral infections. RNase L is a host endoribonuclease and a pivotal component of innate immunity that cleaves viral and cellular RNA within single-stranded loops releasing small structured RNAs with 59-hydroxyl (59-OH) and 39-monophosphoryl (39-p) groups. In 2007, we reported that RNase L cleaves self RNA to produce small RNAs that function as pathogen-associated molecular patterns (PAMPs). However, the precise sequence and structure of PAMP RNAs produced by RNase L is unknown. Here we used hepatitis C virus RNA as substrate to characterize RNase L mediated cleavage products [named suppressor of virus RNA (svRNA)] for their ability to activate RIG-I like receptors (RLR). The NS5B region of HCV RNA was cleaved by RNase L to release an svRNA that bound to RIG-I, displacing its repressor domain and stimulating its ATPase activity while signaling to the IFN-b gene in intact cells. All three of these RIG-I functions were dependent on the presence in svRNA of the 39-p. Furthermore, svRNA suppressed HCV replication in vitro through a mechanism involving IFN production and triggered a RIG-I-dependent hepatic innate immune response in mice. RNase L and OAS (required for its activation) were both expressed in hepatocytes from HCV-infected patients, raising the possibility that the OAS/RNase L pathway might suppress HCV replication in vivo. It is proposed that RNase L mediated cleavage of HCV RNA generates svRNA that activates RIG-I, thus propagating innate immune signaling to the IFN-b gene.

Chimeric poliovirus RNAs, possessing the 5 nontranslated region (NTR) of hepatitis C virus in place of the 5 NTR of poliovirus, were used to examine the role of the poliovirus 5 NTR in viral replication. The chimeric viral RNAs were incubated in cell-free reaction mixtures capable of supporting the sequential translation and replication of poliovirus RNA. Using preinitiation RNA replication complexes formed in these reactions, we demonstrated that the 3 NTR of poliovirus RNA was insufficient, by itself, to recruit the viral replication proteins required for negative-strand RNA synthesis. The 5-terminal cloverleaf of poliovirus RNA was required in cis to form functional preinitiation RNA replication complexes capable of uridylylating VPg and initiating the synthesis of negative-strand RNA. These results are consistent with a model in which the 5-terminal cloverleaf and 3 NTRs of poliovirus RNA interact via temporally dynamic ribonucleoprotein complexes to coordinately mediate and regulate the sequential translation and replication of poliovirus RNA.The single-stranded positive-sense RNA genome of poliovirus functions sequentially as an mRNA for viral protein synthesis and then as a template for viral negative-strand RNA synthesis (33). Cytoplasmic extracts from HeLa cells support the sequential translation and replication of poliovirus RNA (5, 31). In the presence of 2 mM guanidine HCl, cell-free translation-replication reactions support the translation of viral RNA and the accumulation of viral preinitiation RNA replication complexes (4, 6). Guanidine HCl reversibly blocks the initiation of negative-strand RNA synthesis by interfering with the function of viral protein 2CATPase (3,38,40). When preinitiation RNA replication complexes are resuspended in reaction mixtures in the absence of guanidine HCl, the preinitiation RNA replication complexes synchronously initiate the synthesis of viral negative-strand RNA (6). Ribosomes translating the viral mRNA within preinitiation RNA replication complexes prevent the synthesis of negative-strand RNA (8,15). Thus, the conversion of viral ribonucleoprotein complexes into preinitiation RNA replication complexes is an important temporally regulated event in the replication of poliovirus RNA.Contemporary models of eukaryotic mRNA translation suggest that the 5Ј and 3Ј nontranslated regions (NTRs) of mRNAs communicate via RNA binding proteins bound to both termini of the mRNA (14,19,42). In particular, poly(A) binding protein bound to 3Ј-terminal poly(A) interacts with eukaryotic initiation factors eIF4G I and II anchored to the 5Ј NTR of mRNA, bringing the 5Ј and 3Ј NTRs into proximity (49). During the course of a poliovirus infection, viral protein 2APro mediates the cleavage of eIF4G I and II and poly(A) binding proteins (11,18,23,24). This leads to the shutoff of cap-dependent host protein synthesis and precludes the ability of eIF4G I and II and poly(A) binding protein to form proteinprotein bridges between the 5Ј and 3Ј termini of mRNAs, including poliovirus RNA. Therefo...

Chronic hepatitis C virus (HCV) infections are a significant cause of morbidity and mortality worldwide. Interferon-a2b treatment, alone or in combination with ribavirin, eliminates HCV from some patients, but patients infected with HCV genotype 1 viruses are cured less frequently than patients infected with HCV genotype 2 or 3 viruses. We report that HCV mRNA was detected and destroyed by the interferon-regulated antiviral 29-59 oligoadenylate synthetase/ ribonuclease L pathway present in cytoplasmic extracts of HeLa cells. Ribonuclease L cleaved HCV mRNA into fragments 200 to 500 bases in length. Ribonuclease L cleaved HCV mRNA predominately at UA and UU dinucleotides within loops of predicted stem-loop structures. HCV mRNAs from relatively interferon-resistant genotypes (HCV genotypes 1a and 1b) have fewer UA and UU dinucleotides than HCV mRNAs from more interferon-sensitive genotypes (HCV genotypes 2a, 2b, 3a, and 3b). HCV 2a mRNA, with 73 more UA and UU dinucleotides than HCV 1a mRNA, was cleaved by RNase L more readily than HCV 1a mRNA. In patients, HCV 1b mRNAs accumulated silent mutations preferentially at UA and UU dinucleotides during interferon therapy. These results suggest that the sensitivity of HCV infections to interferon therapy may correlate with the efficiency by which RNase L cleaves HCV mRNA.